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Toronto, 500 Sherbourne Street, Toronto, Ontario, Canada M4X lK9. Received 5 March 1976; .... Data are shown for one ratio of effector:target cells only. Recovery of cells in all culture groups at this time. (day 5) was 20+ 6 per cent. ** and tt.
Immunology 1976 31 615

Autoreactivity developing spontaneously in cultured mouse spleen cells II. COMPARISON OF CYTOTOXICITY OF CULTURED MALE AND FEMALE SPLEEN CELLS*

R. M. GORCZYNSKI Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, Ontario, Canada M4X lK9

Received 5 March 1976; acceptedfor publication 18 March 1976

Summary. Male and female spleen cells were compared before and after culture for their cytotoxicity to autologous-embryo fibroblasts. Cultures of male cells developed significantly greater reactivity than cultures of female cells. Moreover, while the cytotoxicity derived from cultures of male cells was totally abolished by treatment of effector cells with a mouse anti-T cell antiserum, such an antiserum had less affect on the effector cells of female mouse cultures.

INTRODUCTION In response to challenge with an allogeneic stimulus whether in vivo or in vitro, mice develop antigenspecific cytotoxic T effector cells. Such effector cells have been quantitated using a 5'Cr cytotoxicity assay (Brunner, Mauel, Rudolf & Chapius, 1970; Miller & Dunkley, 1974) and, more recently, a micro-cytotoxicity assay (Kall & Hellstrom 1975; Takasugi & Klein, 1970). * The author regrets that no reprints of this article will be available. Correspondence: Dr R. M. Gorczynski, Ontario Cancer Institute & Department of Medical Biophysics, University of Toronto, 500 Sherbourne Street, Toronto, Ontario, Canada M4K 1K9.

615

Mouse lymphocytes can also be sensitized to autologous antigens in vitro, with the activity being assayed by a micro-cytotoxicity test (Carnaud, Ilfield, Levo & Trainin, 1974). The previous paper in this series (Gorczynski, 1976) suggested that sensitization to embryo-associated antigens occurred in 5-day cultures of male mouse spleen cells even in the absence of deliberate sensitization with an added source of embryonic antigens. Using a micro-cytotoxicity assay, Carnaud et al. (1974) have investigated the cells responsible for cytotoxicity after auto-sensitization in culture with syngeneic embryo fibroblasts. They found, in keeping with the data from allogeneic stimulation, only evidence for cytotoxic T effector cells. However, these data may be oversimplified in view of the more recent observation that, according to the time of assay in vitro, cytotoxicity or enhanced growth may occur after sensitization to syngeneic or allogeneic antigens (Kall & Hellstrdm, 1975). Recently, Coggin, Ambrose, Bellomy & Anderson (1971) indicated that male and female hamsters did not respond in the same fashion to syngeneic foetal tissue. We have thus investigated whether male and female mouse spleen cells differ in their ability to develop cytotoxic cells in long term (5day) cultures, and whether any such difference can be correlated with qualitative changes in the effector cell types.

R. M. Gorczynski

616

MATERIALS AND METHODS The majority of the techniques used in preceding paper.

were

described

Cell preparation Peritoneal macrophages were prepared from either normal mice (within 36 h of arrival from Cumberland Farms) or from mice given 02 ml incomplete Freund's adjuvant 3 days earlier (Gorczynski, Miller & Phillips, 1973). Irradiation Cells to be irradiated were chilled in ice throughout and given 2000 rad from a 137Cs irradiator. Mice were given a total of 950 rad whole body irradiation. The dose in all cases was 100 rad/minute. Mitogen stimulation assays Stimulation of mouse spleen cells with phytohaemagglutinin (PHA-P, Difco Labs, Detroit, Michigan, U.S.A.) or E. coli lipopolysaccharide (LPS, strain 026: B6, Difco Labs, Detroit, Michigan, U.S.A.) was as described before (Gorczynski, 1974a). Stimulation in this case was measured by [3H]thymidine incorporation at 72 h. Stimulation was expressed as c.p.m. ( +stimulus) c.p.m. (no stimulus).

Antibody-forming-cell (PFC) assays 15 x 106 spleen cells were incubated for 4 days in Marbrook cultures (Marbrook, 1967; Gorczynski, Miller & Phillips, 1971) in a F20 with 2 x 106 sheep erythrocytes (SRBC) (Woodland Farms, Guelph, Ontario), or with 1 pg/ml dinitrophenyl-coupledpolymerized Flagellin (DNP-POL) (a kind gift from Dr M. Feldman). After this time the cells were harvested and antibody-forming-cells (PFC) estimated using a modified Cunningham assay (Cunningham & Szenberg, 1968). Anti DNP-PFC were estimated using TNP-coated SRBC, prepared following the technique of Rittenberg & Pratt (1969). Preparation and testing of heteroantisera All antisera and rabbit complement (mouse spleen cell absorbed) were sterile-filtered through a 0 45 ,um millipore membrane prior to use. All cells to be treated with antiserum were incubated at a cell concentration of 107/ml in PBS at 40 for 60 min. Cells were then washed and resuspended in rabbit complement, diluted 1:10 in a F20 and incubated at 370 for 45 min. The cells were then washed twice and used as required. (i) Hetero anti-T cell antiserum. Rabbit anti-mouse brain theta-associated antigen (anti-BrO) was pre-

Table 1. Specificity of anti-BrO antiserum for murine T lymphocytes Stimulation witht PHA-P

LPS

Treatment of spleen cells*

(1:1000)

100 pg/ml

None

8-4+ 1 9 7-8+1-8 8-2+2-1 1-2+0-2

17-2+ 3-6 19-1+2-3 18-4± 19 22-7+2-3

Anti-BrO Rabbit complement Anti-BrG+rabbit complement

PFC response: SRBC

DNP-POL

(anti-SRBC-PFC) (anti-DNP-PFC) 680+ 94

604+59 696+ 84 42+9

852+ 101 819+68 831+ 92 733+ 194

(974+ 132)§ * Treatment of 6-8 week male C3H spleen cells with antiserum and complement as described in the Material and Methods. Anti-BrO was used at concentration of 1/60. t Mitogen stimulation cultures were set up as described elsewhere (Gorczynski 1974a). 2 x 1O6 spleen cells were used per culture tube. All groups were set up in triplicate. The standard errors shown are computed taking into account the variation in both numerator and denominator. t 15x 106 spleen cells were incubated in Marbrook-type culture vessels and PFC estimated 4 days later as described in the Materials and Methods section. All groups were set up in triplicate. Data are shown as arithmetic means+ standard error of mean. § SRBC-PFC obtained from 15 x 106 (anti BrO+ complement) treated spleen cells+ 2 x 106 SRBCeducated thymus cells (spleen cells taken from irradiated animals given 108 syngeneic thymus cells+ 108 SRBC 6 days before). PFC from 15 x 106 SRBC educated thymus cells alone were 3+2.

Anti-embryo responses of male and female mouse cells

617

Table 2 (a). Specificity of anti-B cell antiserum for murine B lymphocytes assayed in vitro

PHA-P 1:1000

LPS 100 Ug/ml

Irradiated C3H x C57BI/6 spleen cells

SRBC

DNP-POL

Percent specific§ cytotoxicity to 5 x 104 target at 20:1 (effector: target) ratio

7-1+1-6 8-0+ 1l9 6-6+ 1-3

15-1+2-9 16-3+ 1-6 16-2+2-3

5-4+ 1d 5-7+0 7 5 0+0 9

463+59 519+68 522+ 73

601+58 572+75 595+ 63

29+2-2 27+ 1-8 28+2-1

10-2+ 13

2-1+07

6-9+ 1l

94+33

91+22

45+3-1

PFC response:

Stimulation witht Treatment of spleen cells*

None Anti-B serum Rabbit complement Anti-B serum+ rabbit complement

(362± 42)§ (609+ 72)tT *, t and

jAs per Table 1. Anti-B serum was used at a concentration of 1/20. Small numbers (5 x l0) of irradiated

(C3H x C57BL/6) spleen cells were used (fourth column) to stimulate a mixed leucocyte reaction as an additional test of T-cell function. These cells incorporated negligible amounts of 'H-thymidine themselves at 72 h, and were incapable of causing misleading 'back-stimulation' of non-T cells in the responding cell population (first column). § Percent specific cytotoxicity of sensitized C3H cells (to irradiated C57BL/6 spleen cells) was determined at varying effector cell numbers with 5x 104 Con A-Cr51-C57BL/6 spleen cells, as described in the Materials and Methods section. Data are shown for one ratio of effector: target cells only. Recovery of cells in all culture groups at this time (day 5) was 20+ 6 per cent. ** and tt SRBC-PFC responses of 15 x 106 (anti-B serum and rabbit complement) spleen cells in the presence of 5 x 106 bone marrow-B cells (**) or anti-BrO spleen cells (1t) respectively. The SRBC-PFC responses from 15 x 106 of these two latter populations above was 19+4 and 39+ 16.

pared as described by Golub (1971). The specificity of this antiserum for killing (in the presence of rabbit complement) murine T cells and not B cells was established as described earlier (Gorczynski, 1974b) and in Table 1. Thus treatment of spleen cells with this antiserum (at a 1/60 dilution) abolished the mitogen stimulation response to PHA-P and con A, both T-cell mitogens, but not to LPS, a Bcell mitogen. This antiserum also abolished the PFC response of mouse spleen cells to SRBC, a Tdependent antigen, but not to DNP-POL (a Tindependent antigen). The SRBC-PFC response of anti-BrO treated cells was restored by mouse thymocytes educated in vivo to SRBC (Table 1). (ii) Hetero anti-B antiserum (anti-B). Newborn rabbits were injected intraperitoneally (within 2 h of birth) with 1 x 109 mouse thymocytes, and within the first 4 days of birth received three subsequent injections each of 1 x 109 mouse thymocytes. At 10 weeks of age the rabbits were injected intravenously with 2 x 108 adult mouse anti-BrO-treated spleen cells, and similar anti-BrO-treated spleen cell injections were given at 2-weekly intervals for 4 weeks. 10 days atter the last injection the animals

were bled by cardiac puncture, the serum so obtained, heat inactivated (560, 30 min), and frozen at -20° in 2-ml aliquots. Prior to use as an anti-B cell reagent the serum was absorbed three times with one-fifth volume of mouse thymocytes. The data of

Table 2(a) provides data from tissue culture experiments to show that treatment of spleen cells with this antiserum selectively abolished B cell but not T cell functions, i.e. (a) The mitogen response to LPS (but not to PHA or alloantigens) was abolished; (b) The antibody response to DNP-POL and to SRBC was abolished. The response to SRBC was reconstituted with bone marrow B lymphocytes (Gorczynski et al., 1973; Miller & Phillips, 1970), and by anti-BrO-treated spleen cells. (c) The cytotoxic response to mouse alloantigens (as measured by the classical 51Cr cytotoxicity assay, using 5 x 104 5 10 labelled Con A blasts as target cells) was not decreased by treatment with this antiserum. In fact (see Table 2a), the response was generally increased -possible reasons for this will be discussed later. The data of Table 2(b) provides data from in vivo tests of the anti-B cell serum to ensure its functional specificity for B cells rather than macrophages. Since the latter cell is believed to play a

R. M. Gorczynski

618

Table 2(b). Comparison of in vitro and in vivo tests on SRBC and DNP-POL PFC after treatment with anti B-cell antiserum In vitro PPCf Treatment of spleen cells*

SRBC DNP-POL

None 568+84 417+58 Anti-B serum 509+ 68 441+57 Rabbit 535+ 67 407+ 65 complement Anti-B + Rabbit 41+ 6 69+ 17 complement §317+ 41 286+ 33 **30+3 42+ 11 tt27+ 11 30+ 7

In vivo

SRBC

PFCt

DNP-POL

619+84 711+95 694+94 609+42 658+ 90 632+ 59 32+ 5 31+ 16 479+ 69 419+ 33 21+ 11 25+ 19 nd nd

nd = Not done. * Treatment of 6-8 week male C3H spleen cells with antiserum and complement as described in the Materials and Methods section. Anti-B serum was used at a concentration of 1/20. t As per T of Table 1. T 15 x 106 Spleen cells were injected intravenously into irradiated (950 rad) C3H recipients, together with lx 108 SRBC or 10 4ug DNP-POL. PFC were estimated in the spleens of these animals 8 days after transplantation-a minimum of six survivors (per ten mice injected) was seen in all groups. § PFC response of (anti-B +complement) treated spleen cells reconstituted with 5 x 106 anti-Br6 treated spleen cells. The in vitro/in vivo PFC response of the latter cells alone to SRBC-PFC was 12+ 6 and 5+ 5 respectively, and to DNPPOL was 409+ 68 and 371 + 69. ** As per § but using irradiated (1500 rad) anti-Br6 treated cells to reconstitute. The SRBC-PFC from this population alone (in vitro, in vivo) was 2+ 2 and 1 + I respectively, and for DNP-POL was 1 + 1 and 2+ 2. tt As per § and ** but using 5 x 10i irradiated anti-BrO treated peritoneal macrophages to reconstitute the in vitro response of anti-B treated spleen cells. The PFC response of these latter cells alone to SRBC or DNP-POL was 1 + 1 and 0+ 0 respectively.

radio-resistant role in the antibody response to SRBC (Unanue, 1972) and in the cytotoxic response to alloantigens (Wagner, Feldman, Boyle & Schrader 1972), any affect of cytotoxicity to macrophages should become apparent by treating lymphoid cells with the antiserum and then testing their functional capacity in vivo and in vitro (see also below for tests on hetero-anti-macrophage serum). As can be seen from the data of this Table, spleen cells treated with anti-B serum and complement were unable to support an antibody response to SRBC or DNP-POL, whether the response was

generated in vivo or in vitro. This deficiency in the in vitro immune response was not corrected by addition of anti-BrO-treated irradiated peritoneal macrophages. However, the antibody response in vitro and in vivo was reconstituted by radio-sensitive anti-BrO-treated spleen cells. Such synergy in the production of antibody to SRBC in vivo between two inactive populations of radio-sensitive lymphocytes forms the basis of a long-accepted functional description of B and T lymphocytes. Moreover, these reconstitution characteristics (i.e. failure to respond in vivo and in vitro and reconstitution only by sources of B cells) are in marked contrast to data obtained when macrophages are depleted from a lymphoid cell pool (see below).

(iii) Hetero-anti-macrophage antiserum (AMS). Rabbits treated at birth with mouse thymus cells (see above) were injected at 10 weeks with 2 x 101 (anti-BrO-treated) macrophages obtained from the peritoneal cavity of mice given Freund's incomplete adjuvant. Once again this serum was absorbed exhaustively with mouse thymus cells. As described above, one assay for the specific cytotoxicity of this serum to macrophages revolves around the expected difference in immune potential of T and B lymphocytes assayed in vitro (macrophage deficient) or in vivo in irradiated animals (which would still contain functional macrophages). The data of Table 3 indicates that no significant decrease in T (CMI) or B cell responses of treated spleen cells was seen if responses were generated in vivo. However, large reductions in the responses of such cells were seen if the responses were generated in tissue culture. The in vitro response of these treated cells was reconstituted by anti-BrO-treated normal peritoneal macrophages. As can be seen from comparison of Table 2(a) and 2(b) with Table 3, there is little doubt that these antisera are removing quite different cell populations from the responding spleen cell pool. By the functional criteria applied here, and described elsewhere (Gorczynski et al., 1971; Gorczynski et al., 1973) we feel confident in attributing the activities of these two antisera to anti-B cell and anti-macrophage components respectively. RESULTS

Comparison of the ability of male, female and pregnant female mice to show spontaneous cyto-

Anti-embryo responses of male andfemale mouse cells

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Table 3. Specificity of hetero anti-macrophage antiserum In vitro response:

In vivo responset

Treatment of spleen cells* None Anti-macrophage serum (AMS) only Rabbit complement only (AMS+rabbit complement)

SRBC-PFC

51Cr cytotoxicity at 100: 1 (effector: target) ratio

SRBC-PFC

51Cr cytotoxicity at 25: 1 (effector: target) ratio

416+82

16+ 1d

402+39

32+1-7

472+ 93

13+ 2-1

441+ 62

31+ 2-1

563+ 121 511+81

16+ 1-8 17+ 1-3

419+ 31 41+ 11 (377+ 62)§

28+ 1-3 7+0-8 (36+ 1 9)**

* Treatment of 6-8 week, male, C3H, spleen cells was as described in the Materials and Methods section. AMS was used at a final concentration of 1/200. t Irradiated (950 rad) syngeneic mice inoculated intravenously (six mice per group) with 15 x 106 spleen cells and either: (1) 108 SRBC: SRBC-PFC were measured in the spleens of the recipients 8 days later, or (2) 5 x 107 irradiated C57BL/6 spleen cells: specific cytotoxicity was measured in the pooled spleen cells of these recipients 7 days later (as per § of Table 2(a)). I As per () and (d) of Table 2. § and ** SRBC-PFC and cytotoxic responses of 15 x 106 (AMS+ complement) treated spleen cells cultured with 5 x i05 anti-BrO treated irradiated normal peritoneal macrophages. The latter cells alone gave no PFC and no cytotoxicity.

toxicity to autologous embryo fibroblasts after prolonged culture The data in the previous manuscript suggested that male spleen cells, which initially showed no significant cytotoxicity to autologous (or allogeneic) embryo fibroblasts, developed specific cytotoxicity to syngeneic embryo fibroblasts after prolonged culturing for 3-6 days. Since earlier workers had established that male and female animals could respond in a different fashion to embryonic antigen (Coggin et al., 1971), and our earlier data indicated that the cytotoxicity measured was at least in part directed towards embryo-associated antigens, we wondered whether there might be a difference in the spontaneous cytotoxicity developing in cultures of male and female mouse spleen cells. In addition, it was of interest to investigate the cytotoxicity developfrom naturally immunized (to foetal antigens) female mouse cells, i.e. from primiparous females. In this case there was evidence that freshly prepared cells from such animals were already cytotoxic to embryo fibroblasts (Hellstr6m, Hellstrom & Brawn, 1969). Pools of spleen cells were prepared from three age-matched male, female or primiparous female C3H mice. Aliquots of the cells were tested at various dilutions at this time for their cytotoxicity

to C57BL/6-derived embryo fibroblasts (Blef) and C3HIAnf-derived embryo fibroblasts (Cef.). The remainder of the cells were cultured for 5 days in the absence of any stimulus, harvested and were then retested for their cytotoxicity to the same targets. Fresh male spleen cells were also tested at this time to ensure that the targets were not behaving in an unusual fashion. Representative data for one such experiment (of four) are shown in Fig. 1. In keeping with the observation of others, we found that spleen cells from primiparous female mice showed natural cytotoxicity to autologous embryo fibroblasts. Spleen cells from virgin female animals (as already seen for virgin male spleen cells in our earlier work (Gorczynski, 1976)) showed no such cytotoxicity. More interesting, however, was the observation that, after prolonged culture, male spleen cells became naturally cytotoxic for Cef while virgin female cells, and, more dramatically, primiparous female spleen cells, showed much less cytotoxicity. These latter results were particularly surprising since the primiparous female spleen cells were cytotoxic to autologous fibroblasts prior to

culturing. These experiments have been repeated using alternative strain combinations with comparable results.

620

R. M. Gorczynski

4) ca m

10

(a)

t

5 0

20 .Y 0 0

15 0 4, c

a.-

2

10

and cultured as before for 5 days. The cells were harvested under sterile conditions and divided into four equal aliquots. Three were treated with either the anti-BrO (anti-T), anti-B or anti-macrophage serum described earlier (Tables 1-3) and the remainder left untreated. The cells were resuspended to the same volume and tested at various effector: target cell ratios for their cytotoxicity to embryo-fibroblast targets. The data for one experiment (of four) of this type are shown in Fig. 2.

5 0 15

-

( c)

l.Ac

10

7.

5

*0

Ab -.filz v -

-

-

aawIw

2 4 3 5 6 Day of assay (post culture) Figure 1. Comparison of spontaneously occurring cytotoxicity to autologous embryo fibroblasts in long term culture of virgin male (circles) and female (squares) spleen cells and primiparous spleen cell cultures (triangles). Closed symbols represent the cytotoxicity to 3H-labelled embryo fibroblasts prior to culturing, open symbols the cytotoxicity after 5 days in culture. The cytotoxicity of freshly-prepared male spleen cells (xx) at this time of assay (day 5) is also shown. There was no significant difference here (and in subsequent experiments) between the survival rate (17-25 per cent) of virgin male, female or primiparous female spleen cells in culture. All points represent the arithmetic mean (± s.e.) of three cultures. The data of panel (c) was obtained by subtracting that of panel (a) from that of panel (b).

0 .4_ 0

0

Nature of effector cells in cytotoxic populations of male and female mouse spleen cells After deliberate sensitization against embryonic antigens Carnaud et al. have reported that the cytotoxic mechanism is wholely attributed to cytotoxic T effector cells (Carnaud et al., 1974). In view of the different procedures, compared to these authors, we have used to obtain a cytotoxic effector population (in particular no deliberate stimulus was needed in our cultures) we have investigated whether there might also be differences in the effector cells developing in our cultures. We were also interested in potential differences in cytotoxic mechanisms developing in male and female spleen cultures. Spleen cells were pooled from each of three virgin males, females and primiparous females (C3H),

0 4.)

c

CL

5

50:1

500:1 200:1 50:1 500:1 200:1 50:1 500:1 200:1 Effector: target ratio

Figure 2. Effect of treatment with heteroantisera (after 5 days in culture) on the cytotoxicity of cultured male and female C3H spleen cells to autologous embryo fibroblasts. The symbols shown represent treatment with the following sera: V, no treatment; 0, anti-BrO; anti-B; A, AMS. After treatment cells were resuspended to the same volume. The effector: target cell ratios shown are those for untreated cells. (a) Male cells; (b) female cells; (c) primiparous female cells. o,

Once again cultured male cells developed more cytotoxic potential than cultured female cells (either virgin or primiparous). Moreover, marked differences in the effects caused by the respective antisera were seen. No antiserum treatment uncovered significant cytotoxicity in cultures of primiparous female cells. While anti-B serum abolished cytotoxicity from cultures of virgin female cells, this treatment had no affect on male cell cytotoxicity. In contrast, while anti-BrG abolished activity from cultured

Anti-embryo responses of male and female mouse cells

621

Table 4. Effect of treatment with heteroantisera on cytotoxicity of cultured spleen cells to autologous Con A-Cr5l-blast cells Percent

Source of spleen cells* Virgin male

Virgin female

Primiparous female

specific cytotoxicity tot Cr51-C3H Cr51-C57BL/6

Treatmentt None Anti-BrG+ complement Anti-B+ complement AMS+ complement None Anti-BrO+ complement Anti-B+complement AMS+ complement None Anti-BrO+ complement Anti-B+complement AMS+ complement

12-6+ 2-1 32+ 1-0 11-4+ 17 7-3+ 0 9 6-2+ 16 5 9+ 11 2-2+ 1-4 1-8+0-6 2-1+ 1-1 13 + 0-8 1-6+ 1-1 1-4+ 1-3

- 16+ 0 7 10+0 9

0-7+ 12 -1-2+ 1l2 - 11+ 0-9 -0 7+ 11 1-6+ [5 1[0+0-7 -1-3+ 1-6 0 9+ 1 0 -1-4+1-1 0-6+ 0-8

* Spleen cells were pooled from 8 C3H mice of each type and cultured for 5 days. was as described in the Materials and Methods section. All cells were then tested at various ratios with 5 x IO' IICr targets after 12 h. After this time spontaneous release from the targets was 21 + 3-2. The data shown represent an untreated effector cell: target cell ratio of 200: 1. t Arithmetic mean (+ s.e.) of three cultures per point.

t Treatment with the various antisera

male cells, this antiserum had little affect on the cytotoxicity of cultured female cells. Anti-macrophage serum decreased the cytotoxicity seen from both male and female spleen cell cultures, having a generally greater effect on female cells than male cells. Since these data suggested that the cytotoxic effector cells in cultures of male and female spleen cells were quite different, we wondered whether the same result would be obtained if we assayed the cultures in another fashion, (i.e. a 51Cr assay). Accordingly, an analogous experiment to that of Fig. 2 was performed, in which spleen cells were cultured and then treated with various antisera after culture. The cells were then resuspended to the same volume and tested with 5 Cr-labelled Con A spleen cell blasts (syngeneic or allogeneic). The data of Table 4 show the cytotoxicity obtained after 12 h, with a 200: 1 effector: target cell ratio (this represents the ratio with untreated cells). These data support those of Fig. 2, and once again indicate that the cytotoxic effector mechanisms in cultured female spleen cells were not the same as in cultured male spleen cells. Cytotoxicity from the latter was sensitive to anti-BrO and AMS, but not the anti-B, while cytotoxicity from the former was sensitive to anti-B and AMS but not to anti-BrO. H

As a final check to ensure the data of Fig. 2 and Table 4 were not explicable in terms of differences in survival of different cell types in the cultures of the male and female spleen cells used, we have examined the cytotoxic effect (judged by trypan blue exclusion) of the various hetero-antisera for male and female cells before and after culture. The data of Table 5 indicate that by these criteria there are no obvious gross differences in the cellular composition of these populations as judged by specificities detected with these antisera. DISCUSSION In the previous paper of this series we presented data to show that mouse spleen cells left in culture for 5 days developed cytotoxicity to embryoassociated antigens expressed on syngeneic mouse cells. Since many adult human neoplasias express such foetal antigens (Gold & Freedman, 1965; Hollinshead, Glew, Bunnag, Gold & Herberman, 1970) a great deal of interest has recently focused in the immune response of adult cells to such embryonic antigens whether expressed on tumours (e.g. Baldwin, Embleton, Price & Vose, 1974) or embryonic tissue (Carnaud et al., 1971).

R. M. Gorczynski

622

Table 5. Cytotoxicity of Anti-BrO or anti-macrophage serum to virgin male or female cells, or to primiparous female cells, before or after culture for 5 days Percent cytotoxicityt

After culture

Before culture Antiserum used (with rabbit complement)*

Anti-BrO Anti-B serum Anti-macrophage serum *

Virgin female

Virgin male

Primiparous female

Virgin female

Virgin male

Primiparous female

38+ 7 65+9 21+ 6

45+ 6 60+8 15+ 7

39+ 8 71+9 16+ 5

52+ 9 12+5 25+ 10

55+ 9 10+5 27+ 7

50+ 11 15+6 22+ 9

Treatment with antiserum and rabbit complement was as described in the Materials and Methods section.

t Percent cytotoxicity (judged by trypan blue exclusion) for the cell types shown before and after culture

of spleen cell pools made from a minimum of two mice of the type shown. The mean survival of the three cell pools in tissue culture was not significantly different (20+ 5 per cent).

Using an in vivo assay to detect immunity Coggin et al. reported that female and male hamsters responded in different fashions to challenge with embryonic antigens (Coggin et al., 1971). We have seen similar differences in the appearance of spontaneous cytotoxic activity in cultures of virgin male and female mouse spleen cells. Thus male

lymphoid cells developed cytotoxicity after culture whereas female cells showed much less cytotoxicity (Fig. 1, Table 4). Moreover, while primiparous female cells taken directly from pregnant animals were cytotoxic to autologous fibroblasts this reactivity too was greatly diminished after prolonged culture. There are a number of sex differences in the incidence of human neoplasia which may be correlated with such findings as above (Silverberg & Grant, 1970). Recently Parmiani and Lembo suggested that foetal determinants and tumour specific transplantation antigens might evoke qualitatively different responses, with the former perhaps, leading to tumour enhancement (Parmiani & Lembo, 1974).

Using hetero-antisera specific for immune T or B cells or macrophages (see Tables 1-3) we were able to show further differences in the cytotoxicity of cultured male and female cells. The effector cells in cultures of male cells were sensitive to antiBrO (anti-T), and not to anti-B serum while the converse was true for cytotoxic cells in female spleen cell cultures. In both cases, however, an antimacrophage antiserum also markedly decreased the cytotoxicity measured. Moreover, these data were not explainable in terms of different constitution

of the original pre-culture or post-culture spleencell pools (Table 5). Our ability to reach these same conclusions was independent of which of the two assays was adopted (a microcytotoxicity test with adherent targets or a 51Cr assay with suspension culture targets) and further strengthened our belief that this was a physiologically important difference. In addition, the fact that female cells gave a poorer response to autologous cells than male lymphoid cells ruled out the possibility that we were studying an immune response to a sex-linked histocompatibility difference (Billingham, Silvers & Wilson, 1970). These findings were particularly interesting in view of earlier data (Coggin et al., 1971) which suggested that female hamsters could make a good antibody response but a weak (perhaps masked) cellular response to embryonic antigens, while males made a predominantly cell-mediated response to such determinants. Our data could be interpreted in similar terms, with the final effector mechanisms being cytotoxic T cells (Cerottini, Nordin & Brunner, 1970) and activated macrophages (Evans & Alexander, 1972) in the case of male cells, and perhaps antibody facilitated cellular cytotoxicity (MacLennan, Loewi & Harding, 1970) in the case of female cells. Having uncovered a difference in the cytotoxic effector mechanisms of female and male mouse spleen cells to embryonic antigens we were interested in the nature of the cell(s) causing these differences to appear. We have therefore investigated whether by manipulation of the male and female popu-

Anti-embryo responses of male and female mouse cells lations prior to culture we could shed light on the reasons for differences seen in the cytotoxic cell populations present after culture. These experiments are the subject of the following paper.

ACKNOWLEDGMENTS I would like to thank Ms S. Brander for her excellent technical assistance. This work was supported by the Medical Research Council (Grant No. MA5440) and by the National Cancer Institute of

Canada.

REFERENCES BALDWIN R.W., EMBLETON M.J., PRICE M.R. & VOSE B.M.

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